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Research On Gene Engineering Chimeric Antibody Against Feline Parvovirus

Posted on:2019-07-11Degree:MasterType:Thesis
Country:ChinaCandidate:Y J XueFull Text:PDF
GTID:2370330545480290Subject:Veterinary medicine
Abstract/Summary:PDF Full Text Request
Feline parvovirus(FPV),is one of the members of the family Parvoviridae.It can cause pancytopenia in felines,manifested as high fever,stubborn vomiting,diarrhea,dehydration and severe leukocyte reduction,often resulting in feline's infection and high mortality.The current clinical treatment of feline parvovirus and canine parvovirus depends on anti-viral monoclonal antibodies.Mouse monoclonal antibodies are generally not effective in activating the effects of complement and Fc receptors in the treatment of xenogeneic animals and can induce anti-mouse antibodies,resulting in reduced efficacy and aggravating the burden on the animal's immune system.In this study,the recombinant chimeric antibody developed on the canine natural antibody which variable region was replaced by the monoclonal antibody against cat and canine parvovirus,so that the recombinant chimeric antibody retains the high affinity of the monoclonal antibody,to achieve the transformation of canine,and reduce the immunogenicity of antibodies and improve the clinical efficacy of antibodies.In this study,spleen cells and SP2/0 cells were immunized with FPV using PEG method and identified by immunofluorescence(IFA)and neutralizing experiments.The monoclonal antibody 3A8 was obtained.The 3A8 antibody was identified as Neutralizing activity of FPV and CPV.At the same time,the variable region,the position of the constant region,and the secondary structure and partitioning of the natural antibody of canine were studied and analyzed.The variable region gene of the monoclonal antibody and the constant region gene of the canine antibody were cloned,and the genes were chimeric to the recombinant antibody gene.The recombinant antibody gene was cloned into the eukaryotic expression vector pFastBac-Dual baculovirus shuttle vector plasmid,transformed into DH10 Bac competent state,and then extracted through recombinant bacmid DNA and transferred into insect cells for eukaryotic expression.The expressed chimeric antibody was identified by SDS-PAGE,Western-blot,non-denaturing polyacrylamide gel electrophoresis and immunofluorescence experiments.After the above experiments,the recombinant chimeric antibody expressed by the eukaryotic expression of insect cells was confirmed to have a heavy chain size of 55 kDa and a light chain size of 25 kDa.The recombinant chimeric antibody retains its neutralizing activity against FPV and CPV derived from the monoclonal antibody,and its constant region can specifically bind to the anti-dog antibody.This study demonstrated that the modification of the canine source of monoclonal antibodies was achieved by preserving the neutralizing activity of monoclonal antibodies,thereby achieving the goal of reducing the use of antibodies for the immunogenicity of heterologous animals.
Keywords/Search Tags:Feline panleukopenia virus, Monoclonal antibody, Antibody gene area analysis, Genetic engineering antibody
PDF Full Text Request
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